West Nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the MHC II compartment, and enhanced neutralizing antibody response

Virology. 2005 Feb 5;332(1):66-77. doi: 10.1016/j.virol.2004.11.022.

Abstract

A genetic vaccine for West Nile virus (WN) has been synthesized with the WN premembrane-envelope (WN preM-E) gene sequences encoded as a chimera with the transmembrane and carboxyl terminal domains of the lysosome-associated membrane protein (LAMP). The LAMP sequences are used to direct the antigen protein to the major histocompatibility class II (MHC II) vesicular compartment of transfected professional antigen-presenting cells (APCs). Vaccine constructs encoding the native WN preM-E and WN preM-E/LAMP chimera were synthesized in pVAX1 and pITR plasmid backbones. Extracts of human fibroblast 293 and monkey kidney COS-7 cells transfected with the WN preM-E/LAMP chimera constructs contained much greater amounts of E than did the cells transfected with constructs encoding the native WN preM-E. This difference in the concentration of native E and the E/LAMP chimera in transfected cells is attributed to the secretion of native E. The amount of preM protein in cell extracts, in contrast to the E protein, and the levels of DNA and RNA transcripts, did not differ between WN preM-E- and WN preM-E/LAMP-transfected cells. Additionally, confocal and immunoelectron microscopic analyses of transfected B cells showed localization of the WN preM-E/LAMP chimera in vesicular compartments containing endogenous LAMP, MHC II, and H2-M, whereas native viral preM-E lacking the LAMP sequences was distributed within the cellular vesicular network with little LAMP or MHC II association. Mice immunized with a DNA construct expressing the WN preM-E/LAMP antigen induced significant antibody and long-term neutralization titers in contrast to the minimal and short-lived neutralization titer of mice vaccinated with a plasmid expressing the untargeted antigen. These results underscore the utility of LAMP targeting of the WN envelope to the MHC II compartments in the design of a genetic WN vaccine.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Viral / immunology
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Histocompatibility Antigens Class II / immunology*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology*
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / immunology*
  • Vaccines, DNA / genetics
  • Vaccines, DNA / immunology*
  • Vero Cells
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / immunology*
  • West Nile virus / genetics
  • West Nile virus / immunology*
  • West Nile virus / pathogenicity

Substances

  • Antibodies, Viral
  • Histocompatibility Antigens Class II
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Vaccines, DNA
  • Viral Envelope Proteins

Associated data

  • GENBANK/M12294